Recombinant DNA methodologies capable of amplifying purified nucleic acid fragments have long been recognized. Typically, such methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by Cohen et al. (U.S. Pat. No. 4,237,224), Maniatis, T. et al., Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, 1982, etc.
Another method for amplifying a nucleic acid molecule is by template directed extension. By far the most widely used of these methods is the "polymerase chain reaction" ("PCR") (Mullis, K. et al., Cold Spring Harbor Symp. Quant. Biol. 51: 263-273 (1986); Erlich H. et al., EP 50,424; EP 84,796, EP 258,017, EP 237,362; Mullis, K., EP 201,184; Mullis K. et al., U.S. Pat. No. 4,683,202; Erlich, H., U.S. Pat. No. 4,582,788; and Saiki, R. et al., U.S. Pat. No. 4,683,194), which references are incorporated herein by reference) which achieves the amplification of a specific nucleic acid sequence using two oligonucleotide primers complementary to regions of the sequence to be amplified.
The polymerase chain reaction provides a method for selectively increasing the concentration of a nucleic acid molecule having a particular sequence even when that molecule has not been previously purified and is present only in a single copy in a particular sample. The method can be used to amplify either single or double stranded DNA.
The PCR reaction is capable of exponential amplification of specific nucleic acid sequences. Reviews of the polymerase chain reaction are provided by Mullis, K. B. (Cold Spring Harbor Symp. Quant, Biol., 51: 263-273 (1986)); Saiki, R. K., et al. (Bio/Technology 3: 1008-1012 (1985)); and Mullis, K. B., et al. (Met. Enzymol. 155: 335-350 (1987), which references are incorporated herein by reference).
There are many instances in gene cloning and recombinant DNA manipulation in which it is necessary to introduce a primer, probe, or linker sequence into a sample. The above-described nucleic acid amplification procedures are but a small subset of examples.
Often, after a particular reaction step has occurred, the continued presence of the introduced molecule is undesirable. Typically, it has been necessary to physically remove the introduced molecules from the sample. This may be a difficult and complex procedure. Thus, it would be desirable to be able to remove a predefined oligonucleotide from a sample without resorting to physical separation procedures.
In some situations, genetic manipulations have been impeded by the absence of convenient restriction sites, promoters, etc. in a desired orientation with respect to a gene sequence under investigation. It would be desirable to be able to generally alter the gene sequences on either side of a particular, user-defined target sequence.
The present invention provides methods suitable for accomplishing these goals.